Antibodies have been widely accepted for treatment of a variety of diseases, including cancer, arthritis and infectious diseases. Currently more than 300 monoclonal antibody-based drugs are in clinical trials. The predominant advantage of antibody-mediated therapy is its high specificity, facilitated by direct binding to the target(s) for neutralization or elimination (KRAEBER-BODERE et al. 2014). As of Nov. 10, 2014, forty-seven monoclonal antibody products have been approved for medical use in the US or Europe (ECKER et al. 2015). Nevertheless development of antibodies as research reagents, diagnostics and therapeutics with high affinity and specificity remains both time-consuming and labor-intensive. Accordingly an animal-free, high throughput platform for antibody discovery and isolation would accelerate the antibody generation process.
Success in generation of highly specific antibodies in such an ex vivo system depends on the ability to establish highly diverse heavy and/or light chain libraries together with efficient screening capacity e.g., antibody clone identification, validation and antibody production.
One example of a widely used ex vivo system is phage display, in which an antibody fragment (e.g. ScFv) is expressed as a polypeptide fusion to a bacteriophage coat protein and subsequently an antibody of interest is selected by binding to immobilized biotinylated antigen.
As phage display is based on a bacterial protein expression system, it has several shortcomings. For example, some eukaryotic proteins may fold poorly in bacteria, or may require additional post-translational modifications, such as efficient formation of inter- and intra-chain disulfide bonds, which are unavailable in bacterial cells.
In a yeast surface display system, an antibody or antibody fragment (e.g. ScFv, VH, Fab or IgG) is expressed as a polypeptide fusion to a cell membrane anchor protein and subsequently an antibody of interest is selected by binding to immobilized biotinylated antigen in biological panning or by Fluorescence-Activation Cell Sorting or FACS.
In one example of a yeast surface display system, an antibody fragment is fused with yeast 87 amino acid AGA2 membrane protein (Uniprot number P32781, SEQ ID NO:1). Secreted antibody-AGA2 polypeptides are retained on the cell surface thanks to heterodimerization of AGA2 with the yeast membrane anchor protein AGA1 (Uniprot number P32323, SEQ ID NO:2).
To address the lack of protein secretion in eukaryotic display systems, several dual-mode display and secretion systems have been developed, in which cells can produce antibodies that are simultaneously displayed on the cell surface and secreted into the media. The secretion-and-capture system couples antibodies to the host cell surface, e.g., by biotin, where in vivo biotinylated protein of interest is captured onto the avidinated surface of the parent cell (RAKESTRAW et al. 2011). As secreted biotinylated antibodies can bind non-selectively to all avidinated cells, this system exhibits a high incidence of cross-contamination between antibody clones. Similar cross-contamination problems are observed in other systems where the full length secreted antibody is tethered on the host cell surface by binding an immunoglobulin binding protein (RHIEL et al. 2014 and U.S. Pat. No. 9,260,712) that is fused to a cell surface anchor protein.
A dual-mode surface, Fc-bait full antibody display system (SHAHEEN et al. 2013 and U.S. Pat. No. 9,365,846) addresses the cross-contamination problem. In this system, host cells will produce both full IgG antibody and antibody Fc fragment fused with a cell membrane anchor. The latter serves as a bait to capture monovalent antibody fragments. Bivalent IgG antibodies are secreted. As the yeast endoplasmic reticulum (ER), is not equipped for efficient and large scale folding of complex proteins, such as full length human antibodies (DE RUIJTER et al. 2016) the yield of secreted antibody is very low.
Application of the dual-mode surface, Fc-bait full antibody display system to mammalian cell surface display will address the full IgG secretion efficiency issues. Mammalian cell surface systems, however, have several shortcomings, including the cost, experimental time related to mammalian cell cultures as well as the level of antibody library diversity.
Thus, there is a need for an antibody generation system that provides high diversity of antibody repertoire, efficient display of antibodies on the cell surface and an option to secrete antibodies into the media, and that will therefore significantly increase the speed, reduce the cost and improve success of the antibody generation process.
Success in generation of highly specific antibodies in an ex vivo system depends on the ability to establish highly diverse heavy and/or light chain libraries together with efficient screening capacity. Currently ex vivo non-mammalian approaches for generating antibodies such as phage display (HAWKINS et al. 1992), yeast surface display (BODER and WITTRUP 1997; BODER and WITTRUP 2000), ribosome display (HANES and PLUCKTHUN 1997; HE and TAUSSIG 1997), RNA display (REIRSEN et al. 2005), and mammalian cell display (BEERLI et al. 2008) are not intrinsically capable of affinity maturation because they lack the capacity to effect somatic hypermutation. Alternatively, error-prone-PCR followed by labor-intensive sub-library re-cloning steps are generally incorporated into all current ex vivo systems to generate high-affinity antibodies (CHAO et al. 2006 and U.S. Pat. No. 8,691,730). This method is easily doable if the antibody is expressed by a single gene such as in the single-chain variable fragment (scFv) format. When antibodies consist of separate light and heavy chain genes, error-prone PCR sub-libraries have to be constructed for each antigen-specific clone to maintain the correct heavy-light chain pairing. Otherwise random pairing of a light chain from one active antibody with a heavy chain from a different clone will not likely generate again a target-specific antibody.
There is a continuing need in the art for improving the generation of specific antibodies. This invention is to a novel technology platform that combines antibody maturation, cell surface antibody display, and antibody secretion in one system. The invented system can be used for polypeptide library diversification, protein maturation and screening of binder proteins with modified affinity to another molecule. Advantages of this invention include a low cost, rapid growth eukaryotic protein expression and a surface display system with ease of culture and culture maintenance, facile manipulation and genetic engineering. Moreover, yeast mating allows random combination of antibody heavy chain and light chain libraries to form a combined library with highly diverse random H/L combinations, as is known in the art.
In an embodiment of the invention, the expression of lamprey CDA1 (Uniprot number A5H718, SEQ ID NO:3)—the most powerful deaminase mutator of DNA in yeast—which can be in combination with the chemical supermutagen HAP—allows rapid library diversification. In an embodiment of the invention, the expression of any deaminase mutator allows rapid library diversification.
In an embodiment of the invention, an antibody in vivo matured and complexed with membrane anchored bait can be expressed on the surface of the host cell, and/or while instead complexed with a soluble bait the matured antibody can be secreted from the host cell providing effective means for antibody maturation and identification.
In an embodiment of the invention, the use of diploid and/or polyploid yeast strains versus the normally used haploid yeast version protects yeast cells from lethal mutation damage due to the presence of two or more copies of essential genes. With selection methods, such as biological panning, Fluorescence Assisted Cell Sorting (FACS) for cell sorting or ELISA for secreted active antibody validation, yeast cells expressing functional binders can be quickly identified and isolated. Such methods can be used in combination.